Bacteria of the genus Clostridium are gram positive and include many pathogenic species responsible for significant mortality and morbidity in both humans and animals. For instance, Clostridium tetani is a common soil dwelling organism which produces a neurotoxin responsible for the disease tetanus. Clostridium perfringens is a common cause of gas gangrene and food poisoning. Clostridium difficile is a common cause of gastroenteritis and pseudomembraneous colitis, particularly among elderly hospital patients who have had their intestinal flora depopulated by treatment with antibiotics.
Clostridium botulinum is an anaerobic, gram-positive, spore-forming organism that produces the extraordinarily lethal botulinum neurotoxins (BoNTs), a distinctive neurotoxin of extraordinary potency and the cause of botulism, which can cause severe neuroparalytic illness in humans and animals. BoNT is the etiologic agent of botulism, a paralytic disease resulting from the inhibition of neurotransmitter release at the neuromuscular junction. There are several forms of botulism, and infant and foodborne represent the majority of botulism cases reported in the U. S. Despite the unsavory reputation of BoNTs as a deadly poison and as a potential bioterrorism agent, their use in treatment for numerous hyperactive muscle disorders has been widely demonstrated.
Because of their extreme toxicity, the neurotoxins produced by Clostridium botulinum have been the subject of extensive study. Botulinum neurotoxins (BoNT) are classified into seven serotypes, referred to as serotypes A through G, on the basis of their immunological properties. Multiple subtype neurotoxins have been and continue to be discovered especially among serotypes A, B, E and F (Arndt et al. 2006; Carter et al. 2009; Dover et al. 2009; Hill et al. 2007; Smith et al. 2005). In many cases, the amino acid sequences of the toxins have been deduced and compared. See, for example, Minton, “Molecular Genetics of Clostridial Neurotoxins,” in Clostridial Neurotoxins, C. Montecucco (Ed.) Springer-Verlag, Berlin (1995).
Clostridium strains producing BoNTs are broadly characterized into four groups based on metabolic, physiological and genetic properties (Hatheway 1990). Group I contains proteolytic strains of serotypes A, B and F. Group II contains nonproteolytic strains of serotypes B, E and F. Unlike proteolytic strains, nonproteolytic strains lack the ability to digest meat and milk proteins and rely on exogenous proteins for the proteolytic nicking of the neurotoxin into its active di-chain form (Lynt et al. 1982). Group III includes strains of serotypes C and D and Group IV includes strains of C. botulinum serotype G, also referred to as Clostridium argentinense. 
The genes encoding BoNT serotypes C, D and G have long been established to be associated with extrachromosomal elements (Sakaguchi et al. 2005; Zhou et al. 1995). Specifically, the BoNT/C1 and BoNT/D clusters are carried on bacteriophages, and in C. botulinum serotype G, the neurotoxin gene, bont/G, was shown to reside on a large plasmid of ca. 114 kb. However, genes encoding serotypes A, B, E and F were believed to be located on the chromosome. Recently, strains of serotype A, proteolytic and nonproteolytic strains of serotype B, and dual neurotoxin producing Ba, Ab and Bf strains have been shown to harbor neurotoxin genes on very large plasmids (Marshall et al. 2007; Smith et al. 2007; Franciosa et al. 2009). Interestingly, in dual neurotoxin-producing strains of Ba, Ab and Bf subtypes analyzed thus far, it appears that both neurotoxin genes are usually located on the same plasmid. Plasmids identified in proteolytic strains of C. botulinum range in size from approximately 150 to 270 kb and several plasmids found in serotypes A and B and dual neurotoxin producing Ba and Bf strains have been shown to be highly conserved, yet they carry different neurotoxin subtype genes. BoNT-encoding plasmids seem to be more prevalent among strains of serotype B than other serotypes. Unlike the large plasmids observed in proteolytic serotype B strains, plasmids found in nonproteolytic B strains are consistently smaller (approximately 48 kb) and share no homology with plasmids of proteolytic C. botulinum strains
Interest in BoNTs has accelerated due to its potential as pharmaceutical agent for the treatment of segmental movement disorders, spasticity, pain syndromes, and various other neural disorders. In addition, the potential for the use of BoNTs in bioterrorism has been noted and, as a result, government agencies are actively investigating countermeasures against them.
In use, BoNT specifically and tightly binds to cholinergic neurons. BoNT is found natively both in bacterial cultures and in contaminated foods as a progenitor toxin complex in which the neurotoxin is associated with nontoxic components including nontoxic nonhemagglutinin (NTNH), hemagglutinin (HA) proteins, RNA, and other uncharacterized protein components. The neurotoxin component of the toxin complex is a 150 kDa protein comprising a heavy (HC) and a light (LC) chain. The LC contains the catalytic domain that cleaves nerve proteins essential for neurotransmission. Specifically, upon endocytosis and internalization into the nerve terminal, the light chain of the toxin acts to block or slow the exocytotic release of neurotransmitters, particularly acetylcholine. Selective injection of botulinum toxin into neuromuscular regions produces a local weakening of proximal muscles and relief from excessive involuntary muscle contractions. In addition to directly affecting cholinergic neurotransmission, BoNT also exerts other poorly understood effects including altering activity of autonomic ganglia.
Upon endocytosis and internalization into the nerve terminal, the light chain of the toxin acts to block or slow the exocytotic release of neurotransmitters, particularly acetylcholine. Accordingly, the ability of BoNT to specifically target peripheral nerves and its long duration of action make it a very attractive potential therapeutic tool. Complications and drawbacks of botulinum toxin therapy include immunological resistance in some patients and diffusion and resulting apoptosis of neighboring muscles. These side effects can be avoided by proper expression, purification and preparation of the toxin or toxin chains or fragments for pharmaceutical use (Schantz et al. 1992).
BoNT-encoding plasmids carrying neurotoxin genes have been identified in numerous proteolytic and nonproteolytic strains of C. botulinum serotypes A and B and in bivalent subtypes Bf and Ab (Marshall et al. 2007; Smith et al. 2007; Franciosa et al. 2008). Although plasmids among proteolytic strains of C. botulinum are quite large and tend to vary in size, plasmids found in nonproteolytic C. botulinum strains are much smaller and are consistently observed to be approximately 48 kb.
Two main strategies have been utilized to obtain clostridial neurotoxins, individual chains of the toxins, or non-toxigenic components of the toxin complex. The first strategy is to isolate the desired protein itself from cultures of the toxigenic C. botulinum strain, and then biochemically separating the chains. However, separating the chains of the purified toxins, or toxin domains, or toxin fragments is technically challenging, laborious, the yields are low. The clinical use of purified botulinum toxin fragments is thus complicated by the need for extreme purity since even minute amounts of any contaminating active toxin can be non-specific and potentially dangerous. Biochemical preparations of toxin chains or fragments are always contaminated with low levels of active neurotoxin.
The second strategy is to recombinantly produce the toxin or toxin fragments in native or heterologous hosts. Unfortunately, the expression of clostridial genes in most heterologous hosts has been found to be inefficient. Available information on clostridial gene expression in E. coli in particular, and also other heterologous hosts, indicates that the expression of clostridial genes in these hosts occurs at very low levels and is relatively inefficient, and necessary post-translational modifications such as proteolytic activation and molecular folding to not occur. Furthermore, expression of clostridial proteins in heterologous hosts may result in production of degraded product and/or produced as insoluble matter. Expression of clostridial genes in clostridial species is, as might be expected, more efficient and the resulting proteins are less prone to structural or sequence errors and undergo proper posttranslational modifications.
Handling of and culturing of these bacteria is difficult since not only are they highly toxic, the organisms are obligate anaerobes which die if exposed to oxygen. Therefore, the clostridia must be handled under specialized conditions. These technical difficulties reduce efficiency of approaches that can be used for gene transfer in other bacteria such as electroporation, transformation and transduction. For instance, currently used clostridial shuttle vectors are constructed using replication genes from small (less than 10 kb) cryptic clostridial plasmids such as pIP404 (C. perfringens), pCD6 (C. difficile, pCB102 (C. butyricum), pBP1 (C. botulinum) or from small plasmids (2.4-25.5 kb) isolated from E. faecalis (pAMβ1), B. subtilis (pIM13) (Davis et al. 2005; Heap et al. 2009). Besides the replication genes functional in clostridia, these vectors also contain an antibiotic resistance gene functional in both clostridia and E. coli, and these plasmids can be transferred to clostridial strains by electroporation. For instance, vectors that additionally contain E. coli oriT sequences can be introduced into clostridial strains by conjugation from a suitable E. coli donor strain, but there are technical difficulties as described below.
In general, these vectors can be transferred to clostridial strains and are maintained in these strains in the presence of the antibiotic that is encoded from the plasmid vector. Transfer efficiency of these vectors varies and is strain dependent. However, these plasmids are not designed to maintain large gene inserts, e.g. larger than approximately 4 kb. Therefore, they cannot be used for transfer of gene clusters such as botulinum neurotoxin clusters that are 12-16 kb. However, botulinum neurotoxins are naturally produced as protein complexes consisting of a neurotoxin associated with several nontoxigenic components. The complex protects the neurotoxin in the host cell as well as in the human/animal gut. In order to increase the yield and production of high quality botulinum neurotoxins, vectors that can transfer large gene clusters are necessary.
Accordingly, the study and the production of clostridial toxin genes as well as other clostridial genes organized in gene clusters would be greatly facilitated by a plasmid providing the conjugal transfer of BoNT-encoding plasmids in other Clostridium species, thereby providing a plasmid and method for the widespread distribution of BoNT-encoding plasmids in other Clostridium species, especially Clostridium species that do not naturally produce these gene products.